Interleukin-5 antagonist

ABSTRACT

The present invention relates to modified and variant forms of Interleukin-5 molecules capable of antagonizing or reducing the activity of IL-5 and their use in ameliorating, abating or otherwise reducing the aberrant effects caused by native or mutant forms of IL-5.

CROSS REFERENCE TO THE RELATED PATENT APPLICATION

This application is a continuation-in-part application of Ser. No.08/591,438, filed on Apr. 8, 1996 now U.S. Pat. No. 5,939,063.

The present invention relates to modified and variant forms ofInterleukin-5 molecules capable of antagonizing or reducing the activityof IL-5 and their use in ameliorating, abating or otherwise reducing theaberrant effects caused by native or mutant forms of IL-5.

Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and aminoacid sequences referred to in the specification are defined followingthe description.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The rapidly increasing sophistication of recombinant DNA technology isgreatly facilitating research and development in the medical and alliedhealth fields. This is particularly important in the area ofhaemopoietic growth factor research where a number of disease conditionsare predicated on the aberrant effects of native or mutant forms ofgrowth factors.

One particularly important haemopoietic growth factor is IL-5. Thismolecule is a lymphokine secreted by T-cells and mast cells and is adisulfide-linked homodimeric glycoprotein. The human form of thismolecule comprises 114 amino acids per monomer. IL-5 consists of abundle of four a-helices in an up-up, down-down array. The phenomenon ofD-helix swapping whereby one bundle is built up of three helices comingfrom one monomer and a fourth helix which is contributed by the secondmonomer is unique to IL-5. The IL-5 molecule also contains two shortanti-parallel β-strands located between helices A and B and helices Cand D.

Human and murine IL-5 receptors comprise two different chains, the α andβ-subunits.

Human IL-5 binds to the α-subunit but the binding affinity is increasedupon association with the β-chain. The β-chain is shared by othercytokines such as GM-CSF and IL-3.

IL-5 is a haemopoietic growth factor with selectivity for production andactivation of human eosinophils. There is a need, therefore, to developantagonists of IL-5 to act as therapeutic agents for chronic asthma orother disease states with demonstrated eosinophilia or other conditionsassociated with IL-5. It is also important for the IL-5 antagonist notto interfere with the activities of other cytokines, such as GM-CSF orIL-3.

Accordingly, one aspect of the present invention contemplates a modifiedIL-5 comprising a sequence of amino acids within a first a-helix whereinone or more exposed amino acids in said first a-helix having acidic oracidic-like properties are substituted with a basic amino acid residueor a non-acidic amino acid residue.

The IL-5 which is subject to modification is generally of mammalianorigin such as from humans, primates, livestock animals (eg. sheep,cows, pigs, horses), laboratory test animals (eg. mice, rats, guineapigs, rabbits), companion animals (eg. dogs, cats) and captive wildanimals (eg. kangaroos, foxes, deer). Most preferably, the IL-5 is ofhuman origin. The modified IL-5 of the present invention may beglycosylated or unglycosylated and does not interfere with GM-CSF orIL-3 activity.

Even more particularly, the present invention is directed to a modifiedhuman IL-5 molecule comprising a sequence of amino acids wherein Glu atamino acid position 13 (or its equivalent position) in a first a-helixis replaced by Arg or Lys or a chemical equivalent or derivativethereof. An alternative substitution may also be made using non-acidicamino acid residues such as but not limited to Gln and Asn or theirchemical equivalent or derivatives. A “derivative” may be a naturallyoccurring or synthetic amino acid residue.

In accordance with the present invention, it is proposed that themodified IL-5 molecules defined above act as antagonists of the nativeform of IL-5. The term “modified” is considered herein synonymous withterms such as “variant”, “derivative” and “mutant”. The presentinvention is particularly directed to a modified IL-5 which exhibitsspecific antagonism of IL-5 mitogenic effects such as observed in vitro.The modified IL-5 molecules may be glycosylated or unglycosylated and donot interfere with GM-CSF or IL-3 activity.

Accordingly, another aspect of the present invention is directed to anIL-5 antagonist said antagonist comprising an IL-5 molecule having anamino acid sequence in its first at-helix wherein one or more exposedamino acids in said first α-helix having acidic or acidic-likeproperties are substituted with a basic amino acid residue or anon-acidic amino acid residue.

More particularly, the present invention provides an antagonist of IL-5said antagonist comprising an IL-5 molecule with Gln at position 13 (orits equivalent position) in a first α-helix substituted by Arg or Lys ora chemical equivalent or derivative thereof An alternativelysubstitution may also be made using non-acidic amino acid residues suchas but not limited to Gln and Asn or their chemical equivalents orderivatives.

The modified IL-5 molecule of the present invention is preferably inrecombinant or synthetic form and, with the exception of the amino acidsubstitution(s) in the first a-helix, the amino acid sequence of theIL-5 may be the same as the naturally occurring molecule (i.e. nativemolecule) or may carry single or multiple amino acid substitutions,deletions and/or additions to the native amino acid sequence. It is thenreferred to as a “mutant” IL-5. The structure of the first α-helix ofIL-5 has been determined at 2.4 angstrom resolution by X-raycrystallography and comprises amino acid residues 7 to 27 or theirequivalents (see Milburn et al. Nature 363: 172-176, 1993). The modifiedIL-5 of the present invention may or may not comprise a leader sequence.

The nucleotide and corresponding amino acid sequence for the modifiedIL-5 having Arg in position 13 is shown in SEQ ID NOs: 1 and 2 and FIG.1. The leader sequence is shown as amino acids 1 to 6 (Met His Tyr HisHis His [SEQ ID NO:3]). Consequently, amino acids 7 to 27 are shown asamino acids 13-33 in SEQ ID NOs: 1 and 2 and in FIG. 1. Reference toamino acids 7 to 27 is taken as amino acid residue numbers in a moleculewithout a leader sequence. The amino acid sequence for amino acids 7 to27 is shown as SEQ ID NO:4 except that amino acid 13 is represented asXaa. In accordance with the present invention Xaa is preferably a basicamino acid residue or a non-acidic amino acid residue.

Reference to “unglycosylated form” herein means that the molecule iscompletely unglycosylated such as when expressed in recombinant form ina prokaryotic organism (e.g. E. coli). Alternatively, aglycosylation-deficient mammalian cell may be used or completedeglycosylation may occur in vitro using appropriate enzymes. Differentglycosylation patterns are encompassed by the present invention such aswhen recombinant molecules are produced in different mammalian cells.

An “exposed” amino acid is taken herein to refer to an amino acid on asolvent-exposed or outer portion of an α-helix compared to those aminoacids orientated towards the inside of the molecule.

An acidic amino acid includes, for example, Glu and Asp. Preferred basicamino acids are Arg and Lys. Preferred non-acidic amino acids are Glnand Asn.

According to another aspect of the present invention there is provided amodified IL-5 characterised by:

(i) comprising a sequence of amino acids within a first α-helix,

(ii) having one or more exposed amino acids in said α-helix which haveacidic or acidic-like properties being substituted by a basic amino acidresidue or a non-acidic amino acid residue;

(iii) being in recombinant or synthetic form; and

(iv) being capable of antagonising IL-5 mitogenic activity in vitro.

In a related embodiment, the present invention provides an IL-5antagonist characterised by:

(i) comprising a sequence of amino acids within a first α-helix;

(ii) having one or more exposed amino acids in said α-helix which haveacidic or acidic-like properties being substituted by a basic amino acidresidue or a non-acidic amino acid residue;

(iii) being in recombinant or synthetic form; and

(iv) being capable of antagonising IL-5 mitogenic activity in vitro.

The IL-5 mutant may be in glycosylated or unglycosylated form.

This aspect of the present invention is predicated in part on thefinding that a mutation in amino acid 13 (Glu) of human (h) IL-5 to Argresults in the IL-5 variant being capable of antagonising IL-5 mitogeniceffects in vitro. This variant is referred to herein as “IL-5 Arg¹³”“E13 R”. Such a variant would be unable to bind to high affinityreceptors but would still able to fully bind the low affinity a chain ofthe IL-5 receptor. Importantly, the IL-5 Arg¹³ mutant acts as anantagonist, preventing the stimulatory effect of native IL-5. Aparticularly important use of the IL-5 Arg¹³ antagonist is in reducingor otherwise antagonising IL-5-mediated stimulation and activation ofeosinophils in vivo or in vitro. The antagonist may also be able toantagonise effects caused by a mutated, endogenous IL-5. The presentinvention extends to a range of other IL-5 mutants such as IL-5 Lys¹³,IL-5 Gin¹³ and IL-5 Asn¹³ as well as functionally equivalent mutants.The nucleotide and corresponding amino acid sequence for IL-5 Arg¹³(E13R) are shown in SEQ ID NOs: 1 and 2. The present invention extends,in a particularly preferred embodiment, to an isolated polypeptidehaving an amino acid sequence substantially as set forth in SEQ ID NO:2or a genetic sequence encoding same having a nucleotide sequencesubstantially as set forth in SEQ ID NO:1.

By way of a shorthand notation, both single and three letterabbreviations are used for amino acid residues in the subjectspecification and these are defined in Table 1.

Where a specific amino residue is referred to by its position in IL-5, asingle or three letter amino acid abbreviation is used with the residuenumber given in superscript (eg. wherein Xaa^(n), wherein Xaa is theamino acid residue) and “n” is the residue number in the molecule.

TABLE 1 Corresponding Three-letter single-letter Amino acid abbreviationabbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acidAsp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

The present invention is exemplified using IL-5 Arg¹³. This is done,however, with the understanding that the present invention extends toall other IL-5 variants having antagonistic properties to IL-5 in vitroor against eosinophils in vitro or in vivo.

According to another aspect of the present invention there is providedan IL-5 variant comprising an amino acid sequence in the first α-helixof said IL-5:

Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu lie Ala [SEQ ID No. 4];

wherein Xaa is a basic or non-acidic amino acid residue preferablyselected from the group consisting of Arg, Lys, Gln and Asn and whereinsaid variant IL-5 acts as an antagonist for at least one property of thecorresponding native IL-5. The amino acid sequence defined by SEQ IDNO:4 corresponds to amino acid residues 7 to 27 of human IL-5.Preferably, Xaa is Xaa^(n) wherein n is amino acid position 13 of humanIL-5. Preferably Xaa^(n) is Arg¹³ or its equivalent.

In a related embodiment, the present invention contemplates an IL-5antagonist comprising a polypeptide or chemical equivalent thereofcomprising amino acids 7 to 27 of the first α-helix of human IL-5 withthe proviso that one or more exposed amino acids in said first α-helixhaving acidic or acidic-like properties are substituted by a basic aminoacid residue or a non-acidic amino acid residue.

Preferably, the acidic or acidic-like amino acid residue if Glu or Aspand is replaced by Arg, Lys, Gln or Asn.

More preferably, the acidic or acidic-like amino acid residue isreplaced by Arg.

Most preferably, the amino acid sequence of the modified IL-5 is as setforth in SEQ ID NO:2.

In a particularly preferred embodiment, the present invention isdirected to an IL-5 antagonist comprising a modified IL-5 moleculehaving the following amino acid sequence in its first α-helix:

Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO:4]

wherein Xaa is Arg;

or an IL-5 molecule having a single or multiple mutation in its firstα-helix giving functionally similar antagonistic properties to themutation wherein Xaa is Arg.

In respect of the latter embodiment, the mutation in the IL-5 moleculemay be a single or multiple amino acid substitution, deletion and/oraddition or may be an altered glycosylation pattern amongst othermutations. Preferably, the IL-5 antagonist comprises an amino acidsequence as set forth in SEQ ID NO:2.

The IL-5 antagonists of the present invention and in particular IL-5Arg¹³ are useful inter alia in the treatment of allergy, some myeloidleukemias (such as eosinophilic myeloid leukaemia), idiopathiceosinophilic syndrome, allergic inflammations such as asthma, rhinitisand skin allergies by preventing or reducing IL-5-mediated activation ofeosinophils. These and other conditions are considered herein to resultfrom or be facilitated by the aberrant effects of an endogenous nativeIL-5 or an endogenous naturally mutated IL-5.

The present invention, therefore, contemplates a method of treatmentcomprising the administration to a mammal of an effective amount of amodified IL-5 as hereinbefore defined and in particular IL-5 Arg¹³ for atime and under conditions sufficient for effecting said treatment.

Preferably, the treatment is in respect of the treatment of allergy,some myeloid leukemias (such as eosinophilic myeloid leukaemia),idiopathic eosinophilic syndrome, allergic inflammations such as asthma,rhinitis and skin allergies.

Generally, the mammal to be treated is a human, primate, livestockanimal, companion animal or laboratory test animal. Most preferably, themammal is a human.

A single modified IL-5 may be administered or a combination of variantsof the same IL-5. For example, a range of IL-5 variants could be usedsuch as a combination selected from two or more of IL-5 Arg¹³, IL-5Lys¹³, IL-5 Gln¹³ and IL-5 Asn¹³. The IL-5 present invention areparticularly useful in treating eosinophilia and conditions resultingtherefrom such as asthma. Administration is preferably by intravenousadministration but a range of other forms of administration arecontemplated by the present invention including via an implant device orother form allowing sustained release of the IL-5 variant, in anebuliser form or nasal spray. Modified forms of IL-5 permit entryfollowing topical application are also encompassed by the presentinvention.

In addition to the modifications to IL-5 contemplated above, the presentinvention further provides a range of other derivatives of IL-5.

Such derivatives include fragments, parts, portions, mutants, homologuesand analogues of the IL-5 polypeptide and corresponding geneticsequence. Derivatives also include single or multiple amino acidsubstitutions, deletions and/or additions to IL-5 or single or multiplenucleotide substitutions, deletions and/or additions to the geneticsequence encoding IL-5. Derivatives also contemplated modifications toresident nucleotides. Alteration of the nucleotides may result in acorresponding altered amino acid sequence or altered glycosylationpatterns amongst other effects. “Additions” to amino acid sequences ornucleotide sequences include fusions with other peptides, polypeptidesor proteins or fusions to nucleotide sequences. Reference herein to“IL-5” includes reference to all derivatives thereof includingfunctional derivatives or IL-5 immunologically interactive derivatives.All such derivatives would be in addition to the modifications to thefirst α-helix contemplated above. Accordingly, such derivatives would beto IL-5 Arg¹³, IL-5 Lys¹³, IL-5 Gln¹³ or IL-5 Asn¹³.

Analogues of IL-5 contemplated herein include, but are not limited to,modification to side chains, incorporating of unnatural amino acidsand/or their derivatives during peptide, polypeptide or proteinsynthesis and the use of crosslinkers and other methods which imposeconformational constraints on the proteinaceous molecule or theiranalogues.

The present invention further contemplates chemical analogues of IL-5capable of acting as antagonists or agonists of IL-5 or which can act asfunctional analogues of IL-5. Chemical analogues may not necessarily bederived from IL-5 but may share certain conformational similarities.Alternatively, chemical analogues may be specifically designed to mimiccertain physiochemical properties of IL-5. Chemical analogues may bechemically synthesised or may be detected following, for example,natural product screening.

Other derivatives contemplated by the present invention include a rangeof glycosylation variants from a completely unglycosylated molecule to afully glycosylated molecule and from a naturally glycosylated moleculeto molecules with an altered glycosylation pattern. Alteredglycosylation patterns may result from expression of recombinantmolecules in different host cells.

All these types of modifications may be important to stabilise themodified IL-5 molecules if administered to an individual or when used asa diagnostic reagent. The modifications may also add, complement orotherwise facilitate the antagonistic properties of the modified IL-5molecules.

Reference herein to a “modified” IL-5, therefore, includes reference toan IL-5 with an altered amino acid sequence in the first α-helix as wellas, where appropriate, a range of glycosylation variants, amino acidvariations in other parts of the molecule, chemical modifications to themolecule as well as fusion molecules.

The present invention also provides a pharmaceutical compositioncomprising the modified IL-5 molecules as hereinbefore defined orcombinations thereof.

Accordingly, the present invention contemplates a pharmaceuticalcomposition comprising a a modified IL-5, said modified IL-5 comprisinga sequence of amino acids with a first α-helix wherein one or moreexposed amino acids in said first α-helix having acidic or acidic-likeproperties are substituted with a basic amino acid residue or non-acidicamino acid residue, said composition further comprising one or morepharmaceutically acceptable carriers and/or diluents.

In a related embodiment, the present invention provides a pharmaceuticalcomposition comprising a modified human IL-5 or a mammalian homologuethereof said modified IL-5 comprising a sequence of amino acids in afirst α-helix of:

Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO:4]

wherein Xaa is a basic non-acidic amino acid residue preferably selectedfrom Arg, Lys, Gln and Asn, said composition further comprising one ormore pharmaceutically acceptable carriers and/or diluents. Preferably,Xaa is Xaa^(n) where n is amino acid position 13. Preferably, Xaa isArg.

In another related embodiment, the present invention contemplates apharmaceutical composition capable of antagonising IL-5, saidcomposition comprising a modified IL-5 having an amino acid sequence inits first α-helix:

Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO:4]

wherein Xaa is selected from Arg, Lys, Gln and Asn.

Preferably, Xaa is Arg.

The pharmaceutical compositions may also contain other pharmaceuticallyactive molecules including other IL-5 variants. The modified IL-5molecule and other components in a pharmaceutical composition arereferred to below as “active agents” or “active compounds”.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, mannitolglycine or suitable mixtures thereof. The preventions of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thirmerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof. Mannitol glycine is a particularly useful formulationespecially when the modified IL-5 molecules are given as an intravenousdrip.

The present invention also extends to forms suitable for inhaling suchas a nasal spray as well as in nebulizer form. Alternatively, sustainedrelease compositions may be formulated as well as a range of implantdevices. When suitably modified, the molecules may also be given as acream, lotion or gel. A suitable carrier for a cream, lotion or gelincludes a polyol such as but not limited to glycerol, propylene glycol,liquid polyethylene glycol and the like.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, and antibacterial and antifungal agents. Theuse of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, use thereof in thetherapeutic compositions is contemplated by the present invention.Supplementary active ingredients can also be incorporated into thecompositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition involving orfacilitated by aberration of IL-5 molecules or levels of IL-5.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of modulating modified IL-5expression or modified IL-5 activity. The vector may, for example, be aviral vector.

Still another aspect of the present invention is directed to antibodiesto the modified IL-5 molecules and their derivatives. Such antibodiesmay be monoclonal or polyclonal and may be specifically raised tomodified IL-5 or derivatives thereof. In the case of the latter, amodified IL-5 or its derivatives may first need to be associated with acarrier molecule. The antibodies and/or recombinant modified IL-5 or itsderivatives of the present invention are particularly useful astherapeutic or diagnostic agents.

For example, a modified IL-5 and its derivatives can be used to screenfor naturally occurring antibodies to IL-5. These may occur, for examplein some autoimmune diseases. Alternatively, specific antibodies can beused to screen for a modified or mutant IL-5. Techniques for such assaysare well known in the art and include, for example, sandwich assays andELISA. Knowledge of IL-5 levels may be important for diagnosis ofcertain cancers or a predisposition to cancers or for monitoring certaintherapeutic protocols when modified IL-5 molecules are employed.

Antibodies to modified IL-5 of the present invention may be monoclonalor polyclonal. Alternatively, fragments of antibodies may be used suchas Fab fragments. Furthermore, the present invention extends torecombinant and synthetic antibodies and to antibody hybrids. A“synthetic antibody” is considered herein to include fragments andhybrids of antibodies. The antibodies of this aspect of the presentinvention are particularly useful for immunotherapy and may also be usedas a diagnostic tool for assessing apoptosis or monitoring the programof a therapeutic regimen.

For example, specific antibodies can be used to screen for modified IL-5proteins. The latter would be important, for example, as a means forscreening for levels of modified IL-5 in a cell extract or otherbiological fluid or purifying modified IL-5 made by recombinant meansfrom culture supernatant fluid. Techniques for the assays contemplatedherein are known in the art and include, for example, sandwich assaysand ELISA.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies orsynthetic antibodies) directed to the first mentioned antibodiesdiscussed above. Both the first and second antibodies may be used indetection assays or a first antibody may be used with a commerciallyavailable anti-immunoglobulin antibody. An antibody as contemplatedherein includes any antibody specific to any region of modified IL-5.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the enzyme or protein and either type is utilizable forimmunoassays. The methods of obtaining both types of sera are well knownin the art. Polyclonal sera are less preferred but are relatively easilyprepared by injection of a suitable laboratory animal with an effectiveamount of modified IL-5 or antigenic parts thereof, collecting serumfrom the animal, and isolating specific sera by any of the knownimmunoadsorbent techniques. Although antibodies produced by this methodare utilizable in virtually any type of immunoassay, they are generallyless favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art.

Another aspect of the present invention contemplates a method fordetecting modified IL-5 in a biological sample from a subject saidmethod comprising contacting said biological sample with an antibodyspecific for modified IL-5 or its derivatives or homologues for a timeand under conditions sufficient for an antibody-modified IL-5 complex toform, and then detecting said complex.

The present invention also contemplates genetic assays such as involvingPCR analysis to detect a modified IL-5 gene or its derivatives.Alternative methods include direct nucleotide sequencing or mutationscanning such as single stranded conformation polymorphism analysis(SSCP) as well as specific oligonucleotide hybridisation.

The present invention extends to nucleic acid molecules encoding amodified IL-5 of the present invention. Such nucleic acid molecules maybe DNA or RNA. When the nucleic acid molecule is in DNA form, it may begenomic DNA or cDNA. RNA forms of the nucleic acid molecules of thepresent invention are generally mRNA.

Although the nucleic acid molecules of the present invention aregenerally in isolated form, they may be integrated into or ligated to orotherwise fused or associated with other genetic molecules such asvector molecules and in particular expression vector molecules. Vectorsand expression vectors are generally capable of replication and, ifapplicable, expression in one or both of a prokaryotic cell or aeukaryotic cell. Preferably, prokaryotic cells include E. coli, Bacillussp and Pseudomonas sp. Preferred eukaryotic cells include yeast, fungal,mammalian and insect cells.

Accordingly, another aspect of the present invention contemplates agenetic construct comprising a vector portion and a mammalian and moreparticularly a human modified IL-5 gene portion, which modified IL-5gene portion is capable of encoding an modified IL-5 polypeptide or afunctional or immunologically interactive derivative thereof

Preferably, the modified IL-5 gene portion of the genetic construct isoperably linked to a promoter on the vector such that said promoter iscapable of directing expression of said modified IL-5 gene portion in anappropriate cell.

In addition, the modified IL-5 gene portion of the genetic construct maycomprise all or part of the gene fused to another genetic sequence suchas a nucleotide sequence encoding glutathione-S-transferase or partthereof

Accordingly, another aspect of the present invention contemplates anisolated nucleic acid molecule comprising a sequence of nucleotidesencoding or complementary to a sequence encoding a modified IL-5, saidmodified IL-5 comprising a sequence of amino acids with a first α-helixwherein one or more exposed amino acids in said first α-helix havingacidic or acidic-like properties are substituted with a basic amino acidresidue or a non-acidic amino acid residue.

Preferably the sequence of nucleotides encodes a human modified IL-5 ora mammalian homologue which comprises a sequence of amino acids in afirst α-helix of:

Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO:4]

wherein Xaa is a basic or non-acidic amino acid residue preferablyselected from Arg, Lys, Gln and Asn and wherein said modified IL-5exhibits antagonism of IL-5 induced mitogenic effects. Preferably, Xaais Xaa^(n) where n is amino acid position 13. Preferably, Xaa is Arg.

The present invention extends to such genetic constructs and toprokaryotic or eukaryotic cells comprising same.

The present invention further contemplates the use of a modified IL-5 ashereinbefore defined in the manufacture of a medicament in the treatmentof allergy, some myeloid leukemias (such as eosinophilic myeloidleukaemia), idiopathic eosinophilic syndrome, allergic inflammationssuch as asthma, rhinitis and skin allergies.

The present invention is further described by reference to the followingnon-limiting Examples and/or Figures.

In the Figures:

FIG. 1 is (a) a diagrammatic representation of the IL-5 E13R bacterialexpression plasmid pPP31 and (b) DNA/amino acid sequence of MHYH₃-IL-5(E13R). The E13R mutation is circled.

FIG. 2 is a graphical representation showing the titration E13R for itsability to antagonise IL-5-mediated proliferation of TF1.8 cells. IL-5WT IL-5 E13R WT@ 3 ng/ml2+E13R

FIG. 3A shows the titration of E13R for its ability to antagoniseIL-5-mediated proliferation of TF1-8 cells. This figure also shows thatE13R exhibits no detectable agonist activity. IL-5 WT IL-5 E13RIL-5+IL-5 E13R

FIG. 3B shows the failure of E13R to antagonise IL-3-mediatedproliferation of TF1.8 cells. IL-5 IL-5 E13R IL-3+IL-5 E13R

FIG. 3C shows the failure of E 13R to antagonise GM-CSF-mediatedproliferation of TF 1.8 cells. GM IL-5 E13R GM+IL-5E13R

FIG. 4 is a graphical representation showing the titration of IL-3 forits mitogenic effects on TF1.8 cells and the failure of high levels ofE13R to interfere with this activity. IL-5 WT IL-3 E13R@ 100 ng/ml+IL3

FIG. 5 is a graphical representation showing TF 1.8 proliferation assayfor IL-5. IL-5 WT IL-5E13R WT IL5@ 3 ng/ml+E13R WT IL5 (HI)

WT IL-5 (HI) was prepared in inclusion bodies in bacteria, dimerized in2M urea, purified by reverse phase HPLC, concentrated in buffer exchangein PBS. It was never dried down.

IL-5 WT was prepared in the same way as WT IL-5 (HI) but afterconcentration, the preparation was dried down and dissolved in 25 mMglycine and 1.25 mg/ml of mannitol.

FIG. 6 is a graphical representation showing the titration of GM-CSF forits mitogenic effects on TF1.8 cells and the failure of high levels ofE13R to interfere with this activity. IL-5 WT GM E13R@ 1000 ng/ml+GM

FIG. 7 is a graphical representation showing that E13R inhibits IL-5induced eosinophil antibody-dependent cell-mediated cytotoxicity.E13R+IL-5 E13R+GM-CSF

In the absence of GM-CSF and IL-5, the %⁵¹Cr-release was 4%.

FIG. 8 is a graphical representation showing that E13R inhibits IL-5induced eosinophil colony formation. E13R+IL-5 E13R+GM-CSF

No eosinophil colonies were detected absent of GM-CSF and IL-5.

EXAMPLE 1 Production of a Charge-reversal Mutant of Interleukin 5(IL-5)with IL-5-antagonistic Properties

The inventors expressed and purified a charge-reversal mutant of IL-5 inwhich the glutamate residue at position 13 (E13) is replaced by anarginine residue (R) [E13R]. This mutant, E13R, shows specificantagonism of IL-5 mitogenic effects in vitro. In order to maximiseexpression, this protein is presently being expressed as a fusionprotein with the leader sequence MHYH₃ (MHYHHH) [SEQ ID NO:3]. Theleader sequence comprises amino acids 1 to 6 in SEQ ID NOs: 1 and 2.However, there is no reason to believe that removal of this leadersequence (or its replacement by another leader sequence) will affect theantagonistic effects of E13R. A diagrammatic representation of MHYH₃ andthe sequence of E13R is shown in FIG. 1. The nucleotide andcorresponding amino acid sequence of E13R is represented in SEQ ID NOs:1 and 2.

EXAMPLE 2 Production of a Bacterial Expression Plasmid EncodingMHYH₃IL-5 (E13R)

Human IL-5 E13R mutant coding sequence was derived from IL-5 wild-typesequence, by site-specific mutagenesis.

Codon 13 was changed to an Arg codon

CAGCGT

The additional codon changes listed here were based on bacterial codonpreferences to promote translational efficiency in E. coli, and did notlead to amino acid changes.

codon 82: CAACAG

codon 83: AAAAAG

codon 84: AAAAAG

codon 90: AGACGT

codon 91: CGGCGT

codon 92: AGACGT

codon 97: CTACTG

codon 110: GAGGAA

codon 112: ATAATC

codon 113 ATAATC

codon 115 AGTTCC.

The mutant DNA fragment was modified by polymerase chain reaction usingprimers to generate a sequence encoding the leader MHYH₃ at the 5′ end,and a stop codon at the 3′ end. The amplified fragment was subclonedinto the plasmid pEC611 to generate the expression plasmid pPP31,containing the trc promoter.

EXAMPLE 3 Production, Isolation and Refolding of MHYH₃ IL-5 (E13R)

Fermentation is by the Fed-Batch method. Escherichia coliMM294/pACYCLacI^(q) transformed with the E13R-expressing plasmid pPP31is grown in minimal medium at 37° C. The pH is held constant at pH 7.0during bacterial growth by the addition of NH₄OH. Expression of IL-5(E13R) is induced at high cell density (OD₆₀₀>20) either by addition ofIPTG or by feeding with lactose. Induction continues for 5 hours (notincluding lag phase) with the protein being expressed as insolubleinclusion bodies (IBs). Cells are lysed by high pressure homogenization.A primary separation of IBs from cellular debris is done by differentialcentrifugation, after which the IBs are washed and homogenized one moretime. A final centrifugation step isolates IBs of sufficient cleanlinessfor refolding.

The conditions for fermentation, induction, isolation and refolding areas follows:

1. FERMENTATION

A. Inoculation

1. Escherichia coli MM294/pACYClacI^(q) carrying pPP3 1 was streaked outfrom a 80° C. glycerol stock onto a minimal medium plate containingampicillin (100 μg/ml) and kanamycin (30 μg/ml) and grown overnight at37° C.

2. Multiple colonies were assessed for IL-5 (E13R) expression levels bymicroscopic examination of 20 ml shake flask cultures containinginducer, or by colony size on agar plates with or without inducer, inthis instance with IPTG.

3. The selected single colony from an agar plate was transferred into 20ml of nitrogen-rich minimal medium (2) containing amplicillin (100μg/ml)+kanamycin (30 μg/ml). The culture was grown at 37° C. overnightwith agitation.

4. An amount of 10 L of C2 was sterilised in a 22 L fermenter andinoculated to a very low density with overnight culture. Growth was at37° C. and a pH of 7.0 was maintained by the addition of NH₄OH or H₂SO₄.

5. Agitation was manually controlled and aeration automaticallycontrolled with oxygen saturation levels remaining above 10% v/v pO₂.Following depletion of glucose at 16-20 hours the cell mass was fed witha concentrated glucose solution containing additional salts. Nutrientfeed flow rate was determined by pH or oxygen saturated levels.

B. Induction

1. At an optical density of A₆₀₀=>20 the recombinant expression of E13Rantagonist was induced, in this instance by changing to a lactose basednutrient feed. Induction continued for 5 hours at 37° C., pH 7.0.Samples were removed at immediately preceding induction and each hourpost-induction and examined by microscopy for the presence of IB, theirsize being determined by disc centrifugation.

2. The culture was stored overnight at 4° C.

2. PRIMARY ISOLATION OF IL-5 (E13R) INCLUSION BODIES

1. Homogenization—Step 1

The culture was passed five times through a Gaulin 30CD homogenizer at13,500 psi, with homogenate being cooled between passes. Homogenate wasthen diluted with an equal volume of RO H₂O.

The IB size was redetermined by disc centrifugation.

2. Centrifugation—Step 1

The homogenate was centrifuged in a Westfalia SB-7 separator with aconstant speed of 9,210 rpm at a flow rate determined by IB size.

The concentrate collected from this first step was diluted to ≦2.5% w/v.

3. Homogenization—Step 2

The IB suspension was passed once through a Gaulin 30CD homogenizer at13,500 psi.

The IB size was again determined by disc centrifugation.

4. Centrifugation—Step 2

The homogenate was centrifuged in a Westfalia SA-1 separator with aconstant speed of 9,700 rpm at a flow rate determined by IB size.

For refolding, IBs are initially dissolved in 6M Guanidine-HCl,containing 5-10 mM dithiothretiol and buffered to pH 9.5. Reduced IL-5monomers of MHYH₃IL-5(E13R) are then purified from non-reduced proteinby Size Exclusion Chromatography (eg. Superose 12 [Pharmacia]) in 6MGuanidine-HCl, containing 5mM dithiothreitol and buffered to pH 9.5. Thepurified reduced monomer is then refolded into dimers by dilution into2M urea buffered to pH 9.5 at a final protein concentration of 0.01-0.1mg/ml. Purification of correctly folded dimers is by reversed phasechromatography on a High Performance Liquid Chromatograph (HPLC) using asuitable column (eg. Brownlee butyl-silica employing 0.1% v/vtrifluoroacetic acid (TFA) in water as Buffer A and 0.1% v/v TFA inacetonitrile as Buffer B). Purified, correctly folded protein can berecovered in biological buffers after lyophilization from HPLC buffersin the presence of mannitol and glycine, added as bulking excipientagents. The identity of the purified protein can be confirmed using massspectrometry and N-terminal sequencing.

EXAMPLE 4 Assay for IL-5-antagonistic Activity

The biological assay for IL-5 antagonism by E13R uses incorporation ofradiolabelled thymidine to detect IL-5-induced cellular proliferation.The cell line used, TF1.8, is a subline of TF1, a human erythroleukemiccell line which expresses the receptors for IL-3 and GM-CSF. TF1.8differs from TF1 in that it has been selected for expression of the IL-5receptor. Stimulation of TF1.8 proliferation by IL-5 is inhibited by 50%in the presence of a 30-fold excess of E13R, and abolished in thepresence of a 300-fold excess (FIG. 2). However, stimulation of TF1.8proliferation by IL3 or GM-CSF is unaffected by the presence of E13R.These results demonstrate that E13R is a specific and potent antagonistof IL-5. The ³H-thymidine assay was conducted as follows:

Wash cells 24 hrs, prior to assay (1500 rpm/5 min/RT) and resuspend themin cytokine free medium.

Dilute standards and samples to concentrations required, in culturemedium mentioned above (range between 1000 ng/m-1 pg/ml as example).Dilutions were done in triplicate.

Aliquot dilutions of appropriate standards (GM-CSF, IL-3, IL-5, etc.)and samples in 100 ul final volumes, in sterile 96 well, flat bottommicrotitre plates.

Check viability of washed cells and resuspend cells to a concentrationof 5×10⁴ cells/well.

Each cell contains 300 ul of diluted standard/sample+100 ul resuspendedcells.

Plates are incubated @ 37° C. for 72 hrs, in a humidified CO₂ incubator(48 hrs, for TF-1 & TF1.8 cells).

Pulse cells with ³H-Thymidine (1/20 dilution of stock, then add 10ul/well) 0.5 uCl/well.

Incubate plates @ 37° C. for 5 hrs in a humidified CO₂ incubator.

Harvest contents of each well onto filterpaper using cell harvester anddetermine the radioactivity incorporated into the DNA, by liquidscintillation counting.

The results are shown in FIGS. 2 to 6. The results show that E13Rspecifically antagonises IL-5 activity but does not interfere withGM-CSF or IL-3 activity. In particular, the Figures show that E13R doesnot antagonise TF1.8 cell proliferation induced by IL-3 nor does itantagonise TF1.8 cell proliferation induced by GM-CSF.

EXAMPLE 5 hIL-5 Lys¹³

Thr Ser Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO. 5]

EXAMPLE 6 hIL-5 Arg¹³

Thr Ser Ala Leu Val Lys Gln Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO. 6]

EXAMPLE 7 hIL-5 Gln¹³

Thr Ser Ala Leu Val Lys Gln Thr Leu Ala Leu La Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO. 7]

EXAMPLE 8 hIL-5 Asn¹³

Thr Ser Ala La Val Lys Asn Thr Leu Ala Leu Leu Ser Thr His Arg Thr LeuLeu Ile Ala [SEQ ID NO. 8]

EXAMPLE 9 Antagonistic Properties of E13R on Eosinophils

An antibody dependent cell-mediated cytotoxicity (ADCC) assay was usedto determine the effect of E13R on eosinophil function. This assaymeasures the ability of eosinophils to kill target cells radiolabelledwith chromium and expressing a certain antibody recognized by theeosinophils (Vadas et al. J Immunol 130: 795, 1983). Blood was collectedfrom a healthy donor and eosinophils were prepared by metrizamidegradient separation of leukocytes after removal of red cells withdextran (Vadas et al. J Immunol 122: 1228, 1979). The eosinophil purityexceeded 95%. The eosinophils were then incubated with the target cellsat a ratio of 100 eosinophils per target cell, and a fixed dose ofeither GM-CSF (10 ng/ml) or IL-5 (10 ng/ml) and a titration of E13R.FIG. 7 shows that E13R dose-dependently antagonised IL-5-induced ADCC,but had no effect on GM-CSF-induced ADCC. Values are mean and sem oftriplicates.

EXAMPLE 10 Effect of E13R on Proliferation and Differentiation ofEosinophilic Progenitors

Bone marrow was collected from a healthy donor and subjected to densitycentrifugation (Lymphoprep.). The mononuclear cells were plated in agarsupplemented with a fixed dose of either GM-CSF (10 ng/ml) or IL-5 (10ng/ml) and a titration ofE13R. The cells were allowed to grow at 37° C.for 2 weeks. The agar plates were then fixed in gluteraldehyde beforethe plates were stained with Luxol Fast Blue to detect eosinophiliccolonies. FIG. 8 shows that E13R dose-dependently antagonized thecolony-promoting effects of IL-5, while E13R had no effect on GM-CSFstimulation of eosinophilic colony formation. Values are mean and sem oftriplicates.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

8 1 377 DNA nucleotide sequence encoding modified IL-5 CDS (4)..(366) 1cat atg cac tat cac cat cac atc ccc aca gaa att ccc aca agt gca 48 MetHis Tyr His His His Ile Pro Thr Glu Ile Pro Thr Ser Ala 1 5 10 15 ttggtg aaa cgt acc ttg gca ctg ctt tct act cat cga act ctg ctg 96 Leu ValLys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu 20 25 30 ata gccaat gag act ctg agg att cct gtt cct gta cat aaa aat cac 144 Ile Ala AsnGlu Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His 35 40 45 caa ctg tgcact gaa gaa atc ttt cag gga ata ggc aca ctg gag agt 192 Gln Leu Cys ThrGlu Glu Ile Phe Gln Gly Ile Gly Thr Leu Glu Ser 50 55 60 caa act gtg caaggg ggt act gtg gaa aga cta ttc aaa aac ttg tcc 240 Gln Thr Val Gln GlyGly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser 65 70 75 tta ata aag aaa tacatt gac ggc cag aag aag aag tgt gga gaa gaa 288 Leu Ile Lys Lys Tyr IleAsp Gly Gln Lys Lys Lys Cys Gly Glu Glu 80 85 90 95 cgt cgt cgt gta aaccaa ttc ctg gac tac ctg caa gag ttt ctt ggt 336 Arg Arg Arg Val Asn GlnPhe Leu Asp Tyr Leu Gln Glu Phe Leu Gly 100 105 110 gta atg aac acc gaatgg atc atc gaa tcc tgatgaagct t 377 Val Met Asn Thr Glu Trp Ile Ile GluSer 115 120 2 121 PRT modified IL-5 2 Met His Tyr His His His Ile ProThr Glu Ile Pro Thr Ser Ala Leu 1 5 10 15 Val Lys Arg Thr Leu Ala LeuLeu Ser Thr His Arg Thr Leu Leu Ile 20 25 30 Ala Asn Glu Thr Leu Arg IlePro Val Pro Val His Lys Asn His Gln 35 40 45 Leu Cys Thr Glu Glu Ile PheGln Gly Ile Gly Thr Leu Glu Ser Gln 50 55 60 Thr Val Gln Gly Gly Thr ValGlu Arg Leu Phe Lys Asn Leu Ser Leu 65 70 75 80 Ile Lys Lys Tyr Ile AspGly Gln Lys Lys Lys Cys Gly Glu Glu Arg 85 90 95 Arg Arg Val Asn Gln PheLeu Asp Tyr Leu Gln Glu Phe Leu Gly Val 100 105 110 Met Asn Thr Glu TrpIle Ile Glu Ser 115 120 3 6 PRT leader peptide 3 Met His Tyr His His His1 5 4 21 PRT peptide PEPTIDE (7) Xaa can be any amino acid 4 Thr Ser AlaLeu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg 1 5 10 15 Thr LeuLeu Ile Ala 20 5 21 PRT peptide 5 Thr Ser Ala Leu Val Lys Lys Thr LeuAla Leu Leu Ser Thr His Arg 1 5 10 15 Thr Leu Leu Ile Ala 20 6 21 PRTpeptide 6 Thr Ser Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr HisArg 1 5 10 15 Thr Leu Leu Ile Ala 20 7 21 PRT peptide 7 Thr Ser Ala LeuVal Lys Gln Thr Leu Ala Leu Leu Ser Thr His Arg 1 5 10 15 Thr Leu LeuIle Ala 20 8 21 PRT peptide 8 Thr Ser Ala Leu Val Lys Asn Thr Leu AlaLeu Leu Ser Thr His Arg 1 5 10 15 Thr Leu Leu Ile Ala 20

What is claimed is:
 1. An isolated modified interleukin-5 (IL-5)comprising a sequence of amino acids with a first α-helix wherein atleast one of the exposed amino acids in said first α-helix having acidicor acidic-like properties are substituted with a basic amino acidresidue or a non-acidic amino acid residue selected from the groupconsisting of Arg, Lys and Asn, and wherein said modified IL-5 acts asan antagonist of IL-5.
 2. An isolated modified human IL-5 comprising asequence of amino acids in a first α-helix of: Thr Ser Ala Leu Val LysXaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala (SEQ ID NO:4) wherein Xaa is a basic or non-acidic amino acid residue selected fromthe group consisting of Arg, Lys, and Asn and wherein said modified IL-5is an antagonist of IL-5.
 3. An isolated IL-5 antagonist whichantagonizes native human IL-5, comprising a modified IL-5 moleculewherein the amino acid residue at position 13 of the mature portion ofsaid modified IL-5 is a basic amino acid residue or a non-acidic aminoacid residue selected from the group consisting of Arg, Lys, and Asn. 4.An isolated IL-5 antagonist comprising the amino acid sequence as setforth in SEQ ID NO: 2 or the mature portion of SEQ ID NO:
 2. 5. Apharmaceutical composition comprising a modified IL-5, which antagonizesnative human IL-5, said modified IL-5 comprising a sequence of aminoacids with a first α-helix wherein at least one of the exposed aminoacids in said first α-helix having acidic or acidic-like properties aresubstituted with a basic amino acid residue or non-acidic amino acidresidue selected from the group consisting of Arg, Lys and Asn, suadcomposition further comprising at least one of a pharmaceuticallyacceptable carrier or diluent.
 6. A pharmaceutical compositioncomprising (i) a modified human IL-5 which inhibits native human IL-5and comprises a sequence of amino acids in a first α-helix of: Thr SerAla Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu IleAla (SEQ ID NO: 4), wherein Xaa is a basic or non-acidic amino acidresidue selected from the group consisting of Arg, Lys and Asn; and (ii)at least one of a pharmaceutically acceptable carrier or diluent.
 7. Apharmaceutical composition comprising: (i) a modified IL-5 whichantagonizes native human IL-5 and comprises the amino acid sequence asset forth in SEQ ID NO:2 or the mature portion of SEQ ID NO: 2; and (ii)at least one of a pharmaceutically acceptable carrier or diluent.
 8. Thepharmaceutical composition according to claim 6 wherein Xaa of the IL-5is Arg.
 9. The pharmaceutical composition according to claim 6 whereinXaa of the IL-5 is Lys.
 10. The pharmaceutical composition according toclaim 6 wherein Xaa of the IL-5 is Asn.
 11. A method of preventing orreducing IL-5 mediated activation of eosinophils or antibody-dependentcell mediated cytotoxicity (ADCC) in a mammal comprising administeringto said mammal an effective amount of a modified IL-5 for a time andunder conditions sufficient to antagonize a native IL-5 in said mammalthereby preventing or reducing IL-5 mediated activation of eosinophilsor ADCC, said modified IL-5 comprising a sequence of amino acids withina first α-helix wherein at least one of the exposed amino acids in saidfirst α-helix having acidic or acidic-like properties are substitutedwith a basic amino acid residue or a non-acidic amino acid residueselected from the group consisting of Arg, Lys and Asn.
 12. The methodaccording to claim 11 wherein the IL-5 subject to modification is humanIL-5.
 13. A method of preventing or reducing IL-5 mediated activation ofeosinophils or antibody-dependent cell mediated cytotoxicity (ADCC) in amammal comprising administering to said mammal an effective amount of amodified human IL-5 for a time and under conditions sufficient toantagonize a native IL-5 in said mammal thereby preventing or reducingIL-5 mediated activation of eosinophils or ADCC, said modified IL-5comprising a sequence of amino acids in a first α-helix of: Thr Ser AlaLeu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala(SEQ ID NO: 4) wherein Xaa is a basic or non-acidic amino acid residueselected from the group consisting of Arg, Lys, and Asn.
 14. The methodaccording to claim 13 wherein the Xaa of the IL-5 is Arg.
 15. The methodaccording to claim 13 wherein the Xaa of the IL-5 is Lys.
 16. The methodaccording to claim 13 wherein the Xaa of the IL-5 is Asn.